Polyomaviruses have small genomes and their coding capacity is very limited. In order to encode for functions needed in viral DNA replication, transcriptional control, and the manipulation of host functions, polyomaviruses express a series of alternatively spliced forms of their early mRNA that encode T antigens. All polyomaviruses encode a multifunctional large T antigen (LT) and most encode a small T antigen (ST), plus a series of additional poorly characterized early proteins. Of the thirteen polyomaviruses that have been isolated from humans, several express both an LT and ST that appear to be similar in function to those of the well-studied Simian virus 40 (SV40). However, other human polyomaviruses are quite distinct from SV40 regarding both the structure and biological activities of their LT and ST proteins. These include the recently discovered New Jersey polyomavirus (NJPyV) and Human polyomavirus 9 (HuPyV9). In this exploratory application we seek to uncover the unique biology of the NJPyV and HuPyV9 T antigens. Specifically, we will determine whether an alternative T antigen (AT) encoded by NJPyV is in fact an authentic middle T antigen (MT). Thus, far the only polyomaviruses that express a MT protein, murine polyomavirus and hamster polyomavirus, infect rodents. Thus, finding a human virus that expresses a MT would be very novel and suggest unique host manipulation functions. In addition, we will determine how the LT and ST from LPV, a primate polyomavirus closely related to HuPyV9- are able to independently immortalize cells and induce cell proliferation, and will also determine the molecular basis for the robust synergistic action of these proteins. Rather than recapitulate the well-trodden path that has been used to characterize T antigens in the past, we have developed new strategies designed to efficiently identify cellular pathways altered by T antigens from RNA-Seq data, and then to couple this approach with more traditional genetics and proteomics. Using this strategy, we can determine whether HuPyV9 alters the same cellular pathways as SV40 and other similar polyomaviruses, or if this virus manipulates cellular functions by novel mechanisms.